Reproductive BioMedicine Online (2012) 25, 608– 611 www.sciencedirect.com www.rbmonline.com SHORT COMMUNICATION Bisphenol A is not detectable in media or selected contact materials used in IVF Shruthi Mahalingaiah a,b,*,1, Russ Hauser c,d, Donald G Patterson Jr e, Million Woudneh e, Catherine Racowsky a a Department of Obstetrics and Gynecology, Boston Medical Center, Boston, MA, USA; b Boston University, Boston, MA, USA; c Department of Environmental Health, Harvard School of Public Health, Boston, MA, USA; d Vincent Memorial Obstetrics and Gynecology Service, Massachusetts General Hospital, Boston, MA, USA; e AXYS Analytical Services Ltd., Sidney, British Columbia, Canada * Corresponding author. E-mail address: shruthi.mahalingaiah@bmc.org (S Mahalingaiah). 1 Present address: Department of Obstetrics and Gynecology, Boston University Medical Center, 85 East Concord Street, 6th Floor, Boston, MA 02118, USA. Shruthi Mahalingaiah, MD received her medical degree from Harvard Medical School. She completed her residency training in obstetrics and gynaecology followed by fellowship training in reproductive endocrinology and infertility at Brigham and Women’s Hospital. She is currently an assistant professor of obstetrics and gynaecology at the Boston University School of Medicine/Boston University Medical Center. Her area of research interest is environmental influences on reproductive health. Abstract There is a lack of data regarding potential exposure of gametes to bisphenol A during IVF. Detectable concentrations of bisphenol A were not found in commonly used IVF plastic culture dishes, suction tubing or growth media under normal-use conditions. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: bisphenol A, endocrine disruptors, IVF, media, gamete, embryo Introduction Bisphenol A (BPA) is widely used in the production of polycarbonate plastics and epoxy resins and is prevalent in consumer and medical products including those used in direct patient care and for cell culture (Vandenberg et al., 2010). Higher temperature and exposure to acidic and basic solutions can also increase leaching of BPA from plastics, even when complete polymerization has occurred (Vandenberg et al., 2010). BPA has been detected in the urine of men and women undergoing IVF/intracytoplasmic sperm injection (Mahalingaiah et al., 2008) and in follicular fluid at the time of egg retrieval in women undergoing IVF (Ikezuki et al., 2002), suggesting that human gametes may have BPA exposure during gametogenesis. There are preliminary studies evaluating the association of urinary BPA concentrations with IVF outcomes (Fujimoto et al., 2011). The biologically significant lowest dose of BPA exposure has not been as yet determined for the human 1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.08.008 Bisphenol A and IVF supplies gamete or embryo. However, BPA exposure in the early embryonic environment is detrimental to development in mouse models. Studies in 2-cell murine embryo models suggest that concentrations of 1 nmol/l or more had significant effects on embryo development (Takai et al., 2000). In a subsequent study, 2-cell mouse embryos initially cultured in 1 nmol/l BPA and then transferred into the host mother had no differences in birth rate or birthweight, but weight at 21 days was significantly greater compared with those embryos not cultured in 1 nmol/l BPA (Takai et al., 2001). In mouse models, investigators have demonstrated in-utero epigenetic changes to the embryonic genome after maternal dietary exposure to BPA. Methylation changes have been shown as the mechanism of action of BPA on the embryonic genome (Yaoi et al., 2008). Furthermore, these methylation changes have led to demonstrable phenotypic changes which predispose exposed embryos to diseases expressed in neonatal and adult life. These phenotypes range from changes in coat colour, metabolic syndrome, diabetes, adult obesity and behavioural changes (Dolinoy et al., 2007). Because of the ubiquitous presence of BPA in plastics, proprietary information regarding the coatings used in the IVF culture dishes and the lack of data regarding gamete exposure to BPA during the IVF process, this study determined whether BPA is measureable in media used to incubate gametes and in tissue culture plastic products used in an IVF clinic embryology laboratory. Materials and methods The aim was to determine if BPA was present in assisted reproduction media or in the leachate of materials. There were no gametes, embryos or human subjects involved in this study; hence no IRB approval was necessary. Tubing, culture media and culture dishes were tested in common use combinations of the Brigham and Women’s Hospital at the time of this experiment (Table 1) and may not reflect current use conditions. Control aliquots from the same commercial lot of test media were assayed without exposure to tubing or dishes to provide baseline BPA concentrations. Culture media was in its storage container ranging from 52 days to 2.5 months prior to assay in this study. Culture media, plated at the minimum volume needed to evenly coat a test dish (3 ml), was incubated for 48 h at 37°C in 5% CO2 and atmospheric O2. Culture media were then aspirated via glass pipette into glass vials with a silicone-lined polypropylene lid. Suction tubing was loaded with follicle aspiration media and incubated at 37°C for 10 min and transferred into a glass vial. Each combination of media/dish or media control was performed in triplicate. Samples (21 in total) were stored at –20°C until shipped on ice for analysis to AXYS Analytical Services (British Columbia, Canada). The laboratory technicians were blinded to the identity of the samples. At AXYS Analytical Services, samples (1.0–2.5 ml each, were diluted to 5 ml with HPLC water and pH was adjusted to pH 2 with concentrated hydrochloric acid. Each sample was then spiked with 15 ng deuterium-labelled bisphenol A (d6-BPA) and applied to a Waters Oasis glass HLB solid-phase extraction cartridge (200 mg, 5 ml, 60 lm particle). The extract was then reconstituted with 475 ll methanol and 609 spiked with 25 ll of labelled internal standard (equivalent to 15 ng 13C12-BPA). Analysis of sample extracts for BPA was performed in the negative electrospray ionization mode using a Waters 2695 HPLC coupled with a triple quadropole mass spectrometer (Micromass Quattro Ultima MS/MS, LC column, Waters Xterra C18MS, 10.0 cm, 2.1 mm i.d., 3.5 lm particle. Injection volume was 20 ll). On the basis of spiked recovery standards, a ‘specimen detection limit’ was determined for each sample. The method detection limit of each assay was calculated as the greater of two concentrations: (i) the lowest calibration standard converted to a sample equivalent concentration; or (ii) the sample-specific detection limit. All analyte detection limits were 1 ng/ml, and ranged from 0.273 to 0.954 ng/ml (1.197–4.184 nmol/l). Samples were analysed in batches including quality control samples which included: a procedural blank, two spiked reference samples (one lowand one high-level concentration spike) and a reference sample in duplicate using laboratory stock urine for interand intra-batch comparisons. All quality control samples were within specifications for each batch. Results In the media and media/dish combinations tested, no detectable concentrations of BPA were found (Table 1). Therefore, it is unlikely that gametes and embryos are directly exposed to BPA leached from the culture dishes or aspiration tubing tested. Discussion The results of this study suggest that there was no BPA detected in the leachate or media under normal use conditions in human IVF. However, it is unclear whether any exposure (i.e. even below the limit of detection) is of concern in relation to human gametes and embryos. This preliminary study will be useful for future studies exploring the effects of BPA on gametes and embryos since it demonstrates that there is no detectable exposure from commonly used culture dishes and media under normal use conditions. Strengths of this study include that the experiments were set up in triplicate and the reliable method for BPA assessment employed by AXYS Analytical Services, with a sensitive limit of detection. This study is limited in that not all combinations of commercially available media and dish combinations were tested, and the combinations tested in this study are limited to a 48-hour incubation time. Oil overlay was not used in this set of experiments, despite common use of oil overlay. This was due to the issue of clean aspiration of the leachate and the concern over partitioning the BPA into the oil overlay. The comparatively elevated limit of detection of 0.95 ng/ml (4.18 nmol/l) in one analysis batch was due to high laboratory background levels encountered during analysis of the samples in this batch. Although it is reassuring that these results do not demonstrate a detectable exposure of gametes and embryos to BPA, there are many plastic-related chemicals with endocrine activity that may also be in the leachate (Yang 610 S Mahalingaiah et al. Table 1 Culture media and contact materials tested. Colour groupings demonstrate how samples were batched. All samples with the same colour were batched together. Follicle aspiration media (FAM): Dulbecco’s phosphate-buffered saline with phenol red, (Sage, Trumbull, CT, USA), with 5000 IU/ml (Hospira, Lake Forest, IL, USA) added at 0.2 ml heparin per 50 ml. This product was used 2.5 months from date of receipt. Insemination media (IM): Quinn’s Advantage Fertilization (HTF) Medium (Sage) with 5% human serum albumin 100 mg/ml in normal saline (Invitrocare, Frederick, MD, USA). This product was used 52 days after date of manufacture. Embryo culture media (ECM): Vitrolife G1.5v5 medium (Vitrolife, Kungsbacka, Sweden). This product was used 3 months after date of receipt. Dishes 1, 2 and 3 are labelled as polystyrene 6. The suction tubing is labelled as polyethylene 1/2/4. Dish 1: 30–37 Falcon Organ Culture Dish (Becton Dickinson Labware, Franklin Lakes, NJ, USA), used for insemination. Dish 2: 1007 Falcon Dish (Becton Dickinson Labware), used for embryo culture. Dish 3: NUNC 4-well culture dishes (Fischer Scientific, Denmark), used for embryo culture. Suction tubing: Tubing from the Cook ART Echotip Ovum Aspiration Needle with tubing (Cook OB/ GYN, Spencer, Indiana, USA), affixed to a Falcon/Becton Dickinson 2051 Collection Tube (Becton Dickinson Labware). BPA = bisphenol A; LOD = limit of detection; ND = not detected. et al., 2011). Assessing the leachate for a cumulative xeno-oestrogen effect may be more clinically relevant. Future studies are needed to determine the exposure of the gamete and developing embryo as it grows in the in-vitro environment to endocrine disrupting chemicals. Reproductive Biology, Brigham and Women’s Hospital. The authors thank Larisa Altshul for her expertise and Berhan Bogale, embryologist, for his stewardship of the IVF laboratory. References Acknowledgements This work was supported by an Expanding the Boundaries grant from the Department of Obstetrics, Gynecology, and Dolinoy, D.C., Huang, D., Jirtle, R.L., 2007. Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. Proc. Natl. Acad. Sci. USA 104, 13056–13061. Bisphenol A and IVF supplies Fujimoto, V.Y., Kim, D., Vom Saal, F.S., Lamb, J.D., Taylor, J.A., Bloom, M.S., 2011. 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Preimplantation exposure to bisphenol A advances postnatal development. Reprod. Toxicol. 15, 71–74. 611 Vandenberg, L.N., Chahoud, I., Heindel, J.J., Padmanabhan, V., Paumgartten, F.J., Schoenfelder, G., 2010. Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ. Health Perspect. 118, 1055–1070. Yang, C.Z., Yaniger, S.I., Jordan, V.C., Klein, D.J., Bittner, G.D., 2011. Most plastic products release estrogenic chemicals: a potential health problem that can be solved. Environ. Health Perspect. 119, 989–996. Yaoi, T., Itoh, K., Nakamura, K., Ogi, H., Fujiwara, Y., Fushiki, S., 2008. Genome-wide analysis of epigenomic alterations in fetal mouse forebrain after exposure to low doses of bisphenol A. Biochem. Biophys. Res. Commun. 376, 563–567. Declaration: DGP is a consultant for EnviroSolutions Consulting and Principal Scientist for Exponent. The other authors report no financial or commercial conflicts of interest. Brigham and Women’s Hospital does not endorse use of any product discussed in this paper. Received 10 April 2012; refereed 29 June 2012; accepted 29 August 2012.